Relative protein quantification by MS/MS using the tandem mass tag technology.

The determination of protein changes related to stimuli such as pathological conditions is the core task of many proteomic studies. In the past decade, concomitantly to the increasing role of mass spectrometry (MS), several strategies have been implemented for the relative quantification of proteins with MS. Stable isotopic labels are introduced via metabolic, enzymatic, or chemical routes in different samples for their distinction during MS detection. Relative quantification is achieved by comparison of MS or tandem MS (MS/MS) signals of the differentially labeled moieties. Isobaric tagging is an elegant chemical isotope incorporation based on tags with an identical chemical structure and same total mass but with labile parts under collision-activated dissociation, the so-called reporter ions. The reporter ions are characteristic of each tag form and detected at distinct m/z. The TMT, iTRAQ, and ExacTag are examples of such technology. Experimental design, sample preparation and separation, MS acquisition parameters, and data analysis are the key steps to achieve accurate and precise quantitative measurements. We describe herein an isoelectric focusing shotgun proteomics workflow for the relative quantification of proteins in complex mixtures by MS/MS using tandem mass tags.